MRI: Development of Polarization Control for Total Internal Reflection Fluorescence Microscopy

1126312SimonTotal内反射(TIR)荧光显微镜可以在非常低的背景下对膜过程进行高分辨率成像。TIR的独特性质可以用来进行有趣和独特的量化。然而，在对TIR数据进行稳健量化的道路上存在障碍。首先，由于干涉条纹的存在，TIR产生的激发场是不均匀的。这些条纹通常比生物信号的大小更大，是光源、传输光学元件、物镜和样品中的干扰造成的。这使得荧光强度的量化变得困难。控制激发场偏振也是困难的，特别是在校正上面讨论的干涉条纹时。基于极化的TIR可以定量测量蛋白质的水平、取向和动态以及膜的取向。这个小组已经证明，极化可以成为探索分子动力学的强大工具。这项提议的目标是通过重新设计逝去场的照明和建造一台新的显微镜来改进TIR定量成像，以实现：改善TIR激发场的均匀性，控制激发光束，并通过图像采集软件控制激发光束的偏振。

1126312SimonTotal internal reflection (TIR) fluorescence microscopy allows high resolution imaging of membrane processes with very low background. Unique properties of TIR can lend themselves to interesting and unique quantifications. However there are obstacles in the way of robust quantification of TIR data. First, the excitation field created with TIR is non-homogenous due to interference fringes. These fringes, which are often of larger magnitude than the biological signals, result from interferences in the light source, the delivery optics, the objectives and the sample. This makes quantification of fluorescence intensities difficult. It is also difficult to control the excitation field polarization, particularly while correcting for interference fringes discussed above. Polarization based TIR allows for quantitative measurements of the levels, orientation and dynamics of proteins as well as membrane orientations. This group has shown that polarization can be a powerful tool for exploring the dynamics of molecules. The goal of this proposal is to advance quantitative TIR imaging by redesigning the illumination of the evanescent field and building a new microscope to allow: Improved uniformity of the TIR excitation field, control of the excitation beam, and through the image acquisition software control of the polarization of the excitation beam.